Journal: Endocrinology and Metabolism

Article Title: AM1638, a GPR40-Full Agonist, Inhibited Palmitate- Induced ROS Production and Endoplasmic Reticulum Stress, Enhancing HUVEC Viability in an NRF2-Dependent Manner

doi: 10.3803/enm.2023.1774

Figure Lengend Snippet: Fig. 1. Nuclear factor erythroid 2–related factor 2 (NRF2)-mediated signaling was activated after AM1638 (AM) treatment but not after TAK875 (TAK) or LY2922470 (LY) treatment. Human umbilical vein endothelial cells (HUVECs) were stimulated with the indicated G protein-coupled receptor 40 (GPR40) agonists (20 μM) for 24 hours. (A) Kelch-like ECH-associated protein 1 (Keap1), NRF2, and nuclear NRF2 levels were determined by Western blotting. (B) Western blotting shows the intracellular abundance of heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase 1 (NQO1). (C) GPR40 level was limited by small interfering RNA (siRNA). (D, E) In HUVECs transfected with scrambled (Scr) or GPR40 siRNA, nuclear NRF2 level and HO-1 and NQO1 expres- sion were detected by Western blotting. Each mean±standard deviation was obtained from three separate experiments. Veh, vehicle; W.E., whole cell extract; N.E., nuclear extract. aP<0.05 vs. the Veh group; bP<0.05 vs. the AM group; as analyzed by analysis of variance (ANO- VA) followed by the Tukey-Kramer test.

Article Snippet: The GPR40-partial agonists LY2922470 and TAK875 (MedChemExpress, Monmouth Junction, NJ, USA), GPR40-full agonist AM1638 (MedChemExpress), and the NRF2 inhibitor ML385 (Sigma Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Sigma Aldrich).

Techniques: Western Blot, Small Interfering RNA, Transfection, Standard Deviation

Journal: Endocrinology and Metabolism

Article Title: AM1638, a GPR40-Full Agonist, Inhibited Palmitate- Induced ROS Production and Endoplasmic Reticulum Stress, Enhancing HUVEC Viability in an NRF2-Dependent Manner

doi: 10.3803/enm.2023.1774

Figure Lengend Snippet: Fig. 2. Palmitate-induced superoxide production was reduced after AM1638 (AM) treatment but not after TAK875 (TAK) or LY2922470 (LY) treatment. Human umbilical vein endothelial cells (HUVECs) were pretreated with the indicated chemicals for 24 hours and then stim- ulated with palmitate (400 μM) for 6 hours. The cells were stained with dihydroethidium (DHE). (A, C) The superoxide levels were visual- ized using an immunofluorescence microscope (×200). The levels of red fluorescence were obtained using ImageJ software and then nor- malized to the number of cells. (B, D) The mean fluorescence intensity (MFI) for red fluorescence was calculated by flow cytometry. Each mean±standard deviation was obtained from three separate experiments. Veh, vehicle; PA, palmitate; ML, ML385. aP<0.05 vs. the Veh group; bP<0.05 vs. the PA group; cP<0.05 vs. the PA plus AM group, as analyzed by analysis of variance (ANOVA) followed by the Tukey- Kramer test.

Article Snippet: The GPR40-partial agonists LY2922470 and TAK875 (MedChemExpress, Monmouth Junction, NJ, USA), GPR40-full agonist AM1638 (MedChemExpress), and the NRF2 inhibitor ML385 (Sigma Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Sigma Aldrich).

Techniques: Staining, Immunofluorescence, Microscopy, Fluorescence, Software, Flow Cytometry, Standard Deviation

Journal: Endocrinology and Metabolism

Article Title: AM1638, a GPR40-Full Agonist, Inhibited Palmitate- Induced ROS Production and Endoplasmic Reticulum Stress, Enhancing HUVEC Viability in an NRF2-Dependent Manner

doi: 10.3803/enm.2023.1774

Figure Lengend Snippet: Fig. 3. Palmitate-induced endoplasmic reticulum (ER) stress was inhibited after treatment with AM1638 (AM), TAK875 (TAK), or LY29 22470 (LY). (A, B, C) Human umbilical vein endothelial cells (HUVECs) were pretreated with the indicated G protein-coupled receptor 40 (GPR40) agonist (20 μM) for 24 hours and then stimulated with palmitate (400 μM) for 6 hours. Western blotting showed the intracellular levels of Kelch-like ECH-associated protein 1 (Keap1), heme oxygenase-1 (HO-1), protein kinase R-like endoplasmic reticulum kinase (PERK) phosphorylation, CCAAT/enhancer‐binding protein homologous protein (CHOP), inositol requiring enzyme 1α (IRE1α) phosphor- ylation, and spliced X-box binding protein-1 (XBP-1s). (D, E, F) HUVECs were pretreated with AM1638 (20 μM) or AM1638 plus ML385 (2 or 5 μM) for 24 hours and then incubated with palmitate (400 μM) for 6 hours. The levels of the indicated proteins were determined by Western blotting. Each mean±standard deviation was obtained from three separate experiments. Veh, vehicle; PA, palmitate; ML, ML385. aP<0.05 vs. the Veh group; bP<0.05 vs. the PA group; cP<0.05 vs. the PA plus AM group, as analyzed by analysis of variance (ANOVA) followed by the Tukey-Kramer test.

Article Snippet: The GPR40-partial agonists LY2922470 and TAK875 (MedChemExpress, Monmouth Junction, NJ, USA), GPR40-full agonist AM1638 (MedChemExpress), and the NRF2 inhibitor ML385 (Sigma Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Sigma Aldrich).

Techniques: Western Blot, Phospho-proteomics, Binding Assay, Incubation, Standard Deviation

Journal: Endocrinology and Metabolism

Article Title: AM1638, a GPR40-Full Agonist, Inhibited Palmitate- Induced ROS Production and Endoplasmic Reticulum Stress, Enhancing HUVEC Viability in an NRF2-Dependent Manner

doi: 10.3803/enm.2023.1774

Figure Lengend Snippet: Fig. 4. Palmitate-induced cell death was blocked after treatment with AM1638 (AM), TAK875 (TAK), or LY2922470 (LY). Human umbili- cal vein endothelial cells (HUVECs) were pretreated with the indicated chemicals for 24 hours and then stimulated with palmitate (400 μM) for 24 hours. (A, C) Western blotting showed the cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP) levels. (B, D) Cell viability was measured using EZ-CYTOX solution (Daeil Lab Service). Each mean±standard deviation was obtained from three separate experiments. Veh, vehicle; PA, palmitate; ML, ML385; OD, optical density. aP<0.05 vs. the Veh group; bP<0.05 vs. the PA group; cP<0.05 vs. the PA plus AM group, as analyzed by analysis of variance (ANOVA) followed by the Tukey-Kramer test.

Article Snippet: The GPR40-partial agonists LY2922470 and TAK875 (MedChemExpress, Monmouth Junction, NJ, USA), GPR40-full agonist AM1638 (MedChemExpress), and the NRF2 inhibitor ML385 (Sigma Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Sigma Aldrich).

Techniques: Western Blot, Standard Deviation

Journal: Endocrinology and Metabolism

Article Title: AM1638, a GPR40-Full Agonist, Inhibited Palmitate- Induced ROS Production and Endoplasmic Reticulum Stress, Enhancing HUVEC Viability in an NRF2-Dependent Manner

doi: 10.3803/enm.2023.1774

Figure Lengend Snippet: Fig. 5. Palmitate-induced nuclear damage was reduced after treatment with AM1638 (AM), TAK875 (TAK), or LY2922470 (LY). Cells were pre-incubated with the indicated chemicals for 24 hours and then stimulated with palmitate (400 μM) for 24 hours. (A, C) Human um- bilical vein endothelial cells (HUVECs) were stained with Hoechst to observe the nuclear morphology under a fluorescence microscope (×200). The white arrows point to cells whose nucleus changed. (B, D) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive and -negative HUVECs were counted under a fluorescence microscope (×200). The white arrows indicate nuclear- cleaved cells. Each mean±standard deviation was obtained from three separate experiments. Veh, vehicle; PA, palmitate; ML, ML385; DAPI, 4´,6-diamidino-2-phenylindole. aP<0.05 vs. the Veh group; bP<0.05 vs. the PA group; cP<0.05 vs. the PA plus AM group, as ana- lyzed by analysis of variance (ANOVA) followed by the Tukey-Kramer test.

Article Snippet: The GPR40-partial agonists LY2922470 and TAK875 (MedChemExpress, Monmouth Junction, NJ, USA), GPR40-full agonist AM1638 (MedChemExpress), and the NRF2 inhibitor ML385 (Sigma Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Sigma Aldrich).

Techniques: Incubation, Staining, Fluorescence, Microscopy, TUNEL Assay, Standard Deviation

Journal: Endocrinology and Metabolism

Article Title: AM1638, a GPR40-Full Agonist, Inhibited Palmitate- Induced ROS Production and Endoplasmic Reticulum Stress, Enhancing HUVEC Viability in an NRF2-Dependent Manner

doi: 10.3803/enm.2023.1774

Figure Lengend Snippet: Fig. 6. The actions of G protein-coupled receptor 40 (GPR40)-spe- cific agonists in human umbilical vein endothelial cells. AM1638 (AM), a GPR40-full agonist, can activate nuclear factor erythroid 2– related factor 2 (NRF2)-related pathways and reduce palmitate-in- duced superoxide production, enhancing cell viability. However, GPR40-partial agonists, TAK875 (TAK) and LY2922470 (LY), can decrease palmitate-induced cytotoxicity without blocking superox- ide production or activating NRF2 pathways. HO-1, heme oxygen- ase-1; NQO1, nicotinamide adenine dinucleotide phosphate: qui- none oxidoreductase 1; ER, endoplasmic reticulum; PERK, protein kinase R-like endoplasmic reticulum kinase; CHOP, CCAAT/en- hancer‐binding protein homologous protein; IRE1α, inositol requir- ing enzyme 1α; XBP-1, X-box binding protein-1; PARP, poly (ADP- ribose) polymerase.

Article Snippet: The GPR40-partial agonists LY2922470 and TAK875 (MedChemExpress, Monmouth Junction, NJ, USA), GPR40-full agonist AM1638 (MedChemExpress), and the NRF2 inhibitor ML385 (Sigma Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Sigma Aldrich).

Techniques: Blocking Assay, Binding Assay

Journal: Cancer Science

Article Title: Peripubertal high‐fat diet promotes c‐Myc stabilization in mammary gland epithelium

doi: 10.1111/cas.14492

Figure Lengend Snippet: Oleic acid (OA) stabilizes c‐Myc in mouse mammary epithelial cells (MMECs). A, Serum nonesterified fatty acid (NEFA) concentration in mice fed with control diet (CD) or high‐fat diet (HFD) for 5 weeks (N = 3). B, Immunoblotting (IB) of indicated proteins in CD‐MMECs treated with BSA or 0.15 mmol/L BSA‐conjugated OA for 48 h (N = 4). C, Cycloheximide (CHX) assay of CD‐MMECs treated with BSA or 0.15 mmol/L BSA‐conjugated OA for 48 h followed by 10 µg/mL CHX treatment for the indicated time periods (N = 3). D, Quantitative analysis of c‐Myc half‐life in CD‐MMECs and HFD‐MMECs (N = 3). E, Colony formation assay of CD‐MMECs (N = 3). Cells were treated with BSA or 0.15 mmol/L BSA‐conjugated OA . Cells (4 × 10 3 ) were seeded on day 0 onto 60‐mm dishes and stained with crystal violet on day 14. F, Quantitative analysis of the colony area in (E). G, Mammosphere formation assay of CD‐MMECs treated with BSA or 0.15 mmol/L BSA‐conjugated OA (N = 3). H, Quantitative analysis of the sphere number in (G). Scale bar = 100 µm (upper), 200 µm (bottom). I, IB of indicated proteins in CD‐MMECs treated with BSA or 0.15 mmol/L BSA‐conjugated OA followed by treatment with DMSO or 50 nmol/L U0126 for 1 h. J, Colony formation assay of CD‐MMECs treated with BSA or 0.15 mmol/L BSA‐conjugated OA for 10 days and then with DMSO or 50 nmol/L U0126 for additional 7 days. Cells (6 × 10 3 ) were seeded on day 0 onto 60‐mm dishes and stained with crystal violet on day 14 (N = 3 for each group). Cell culture medium was replenished every 72 h. K, Quantitative analysis of the colony area in (J) (N = 3 each group). L, IB of indicated proteins in CD‐MMECs treated with DMSO or 100 nmol/L TAK‐875 (G protein‐coupled receptor [GPR40] agonist) for indicated time periods. M, IB of indicated proteins in CD‐MMECs treated with DMSO or 100 nmol/L GPR40 agonist TAK‐875 for 24 h and then with DMSO or 50 nmol/L U0126 for additional 1 h. α‐Tubulin was used as a loading control. Cells were cultured in serum‐free complete EpiCult‐B medium supplemented with recombinant human (rh) basic fibroblast growth factor, rh epidermal growth factor, heparin, and insulin. Data are mean ± SE, * P < .05, ** P < .01, *** P < .001 (Student’s t test and 2‐way ANOVA)

Article Snippet: Mouse mammary epithelial cells were cultured in FBS‐free complete EpiCult‐B medium (Mouse) (#05610; STEMCELL Technologies) as described above and treated with 100 nmol/L GPR40 agonist (TAK‐875; ChemScene) or DMSO (Nacalai Tesque).

Techniques: Concentration Assay, Control, Western Blot, Colony Assay, Staining, Tube Formation Assay, Cell Culture, Recombinant